This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We report here a second case of coreceptor switch in R5 simian-human immunodeficiency virus SF162P3N (SHIV(SF162P3N))-infected macaque CA28, supporting the use of this experimental system to examine factors that drive the change in coreceptor preference in vivo. Virus recovered from CA28 plasma (SHIV(CA28NP)) used both CCR5 and CXCR4 for entry, but the virus recovered from lymph node (SHIV(CA28NL)) used CXCR4 almost exclusively. Sequence and functional analyses showed that mutations in the V3 loop that conferred CXCR4 usage in macaque CA28 differed from those described in the previously reported case, demonstrating divergent mutational pathways for change in the coreceptor preference of the R5 SHIV(SF162P3N) isolate in vivo.